The following description of the background of the invention is provided simply as an aid in understanding the invention and is not admitted to describe or constitute prior art to the invention.
Angiotensin converting enzyme (ACE) is a zinc metalloproteinase involved in the renin-angiotensin and in the kallikrein-kinin systems, in which it is responsible for the proteolytic activation of angiotensin I and bradykinin. Because of the central role played by the renin-angiotensin and kallikrein-kinin systems in regulating blood pressure and electrolyte balance, ACE has been identified as an important therapeutic target for diseases such as essential hypertension, diabetic neuropathy, renal disease, congestive cardiomyopathies including congestive heart failure, and myocardial infarction. See, e.g., Cambien et al., Nature 359: 641-44 (1992); U.S. Pat. No. 5,359,045; Higaki et al., Circulation 101: 2060-65 (2000); Kennon et al., Diabet. Med. 16: 448-58 (1999). ACE has also been identified as a risk factor for stent restenosis following treatment for coronary artery disease. See, e.g., Ribichini et al., Circulation 97: 147-54 (1998).
ACE is mainly located on the endothelium of blood vessels, especially in the pulmonary circulation, but it is also found in epithelial cells, in blood mononuclear cells, in macrophages, in male germinal cells and in a circulating form in several biological fluids. Circulating ACE probably originates from the vascular endothelial cells. In plasma and on the surface of endothelial cells, ACE converts the inactive decapeptide angiotensin I into the highly vasoactive and aldosterone-stimulating octapeptide angiotensin II. Angiotensin II is a powerful vasoconstrictor which may modulate or induce the growth of vascular smooth muscle cells and cardiomyocytes. ACE can affect the oxidation of low density lipoproteins (LDLs), endothelial cell function, and smooth muscle cell migration and proliferation, which are all important components of atherosclerosis.
The human ACE gene is located on chromosome 17q23 and includes 26 exons. Its coding sequence is 4.3 kb in length and codes for a protein of 1,306 amino acids. The ACE gene is present in the population as different allelic variants. A variant of particular interest clinically is the presence or absence of a 287 base pair (“bp”) non-coding fragment within Intron 16. When this 287 bp sequence is present in an ACE gene, the genotype is designated “I” for “insertion”; conversely, when this 287 bp sequence is absent in an ACE gene, the genotype is designated “D” for “deletion.” Because the genome contains two copies of each gene, referred to as “alleles,” possible ACE genotypes with regard to this variant are D/D, I/D, and I/I.
Increased ACE activity correlates strongly with the deletion/deletion (D/D) and insertion/deletion (I/D) genotypes. The D/D genotype has also been associated with myocardial infarction, ischemic and idiopathic dilated cardiomyopathy, sudden death in hypertrophic cardiomyopathy, and restenosis after percutaneous transluminal coronary angioplasty. In addition, an increased risk of coronary artery disease is attributed to the ACE D/D genotype. The ACE genotype of an individual has also been related to response to ACE inhibitors (such as benazepril, captopril, cilazapril, enalapril, enalaprilat, fosinopril, lisinopril, moexipril, perindopril, quinapril, ramipril, and trandolapril) and to angiotensin II type 1 receptor antagonists (such as irbesartan, losartan, valsartan, telmisartan, camdesartam, and eprosartan). See, e.g., Kurland et al., J. Hypertens. 19: 1783-87 (2001); Okumura et al., Circ. J. 66: 311-16 (2002).
Polymerase chain reaction (“PCR”) amplification, followed by agarose gel electrophoresis, is commonly used to identify the ACE genotype present in a sample. It has been reported, however, that such PCR methods result in significant mistyping. See, e.g., Odawara et al., Hum. Genet. 100: 163-66 (1997); Shanmugan et al., PCR Methods Applications 3: 120-21 (1993); Rigat et al., Nucl. Acid Res. 20: 1433 (1992). To eliminate the mistyping, a second PCR reaction that detects only the I/I and I/D genotypes is typically performed to confirm the D/D genotype. Since only the I/I and I/D genotypes can be detected in the second reaction, the absence of a PCR fragment is taken as indicating a true D/D genotype. Such methods, however, cannot distinguish an unsuccessful PCR reaction from a true D/D genotype.
Each publication and patent in the foregoing section is hereby incorporated by reference in its entirety, including all tables, figures, and claims.